Previously, we showed that a unique genotype 2a JFH-1 genome replicates efficiently and secrets viral particles after transfection into a human hepatoma cell line. The viral particles have a density of about 1.17 gml with an average diameter of about 55 nm. The secreted virus is infectious for Huh7 cells. The cell culture-generated HCV is infectious in chimpanzee. [unreadable] [unreadable] Recently, we also showed the production of HCV particles by using a DNA expression plasmid containing full-length HCV cDNA flanked by self-cleaving ribozymes. This construct also contains the secreted alkaline phosphatase gene to monitor the expression from this construct and to normalize transfection efficiency and to control for effects of culture conditions, such as anti-viral testing. We produced HCV particles of various genotypes including 1a (H77), 1b (CG1b) and 2a (J6 and JFH-1) in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and effects of culture conditions. After transfection into Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells were inoculated to naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had identical sequences as the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.[unreadable] [unreadable] Current treatment of chronic hepatitis C based on combination of peginterferon and ribavirin is only effective in about half of the patients and is accompanied by substantial side effects. Developing new classes of drugs against HCV is crucial. Phosphorothioate oligonucleotides (PS-ONs) have a sequence-independent antiviral activity against HIV-1 by inhibiting virus-cell fusion. Because viral entry is a highly conserved mechanism, this antiviral action of PS-ONs may be effective against infection by other enveloped viruses with type I or II fusion mechanisms. By applying the cell culture system described above, we assessed whether PS-ONs inhibit HCV infection and to evaluate the antiviral mechanism of action of PS-ONs. Various forms of PS-ONs and the control phosphodiester oligonucleotides (PO-ONs) were synthesized and evaluated in infectious HCV cell culture system systems. To test the efficacy of PS-ON in vivo, human hepatocytes transplanted uPASCID mice were inoculated with infectious HCV and treated with the PS-ON. The PS-ONs exhibited potent inhibitory activities in both cell culture-generated HCV-JFH1 (HCVcc) and HCV pseudo-particles (HCVpp) systems. This inhibitory activity was size and phosphorothioation dependent but sequence independent. The control PO-ONs had no inhibitory activity against HCV infection. The PS-ONs had no effect on viral replication in the HCV replicon system and binding of HCV-LPs to cells, indicating that the target of inhibition by PS-ONs is at the post-binding, cell-entry step. In human hepatocyte-engrafted uPASCID mice, the PS-ONs also appeared to efficiently block de novo HCV infection. The PS-ONs (amphipathic DNA polymers) are a promising new class of antiviral compounds that inhibit HCV fusion and entry.